Calf diarrhea virus vaccine and processes

ABSTRACT

1. AN INACTIVATED REOVIRUS-LIKE CALF-DIARRHEA VIRUS VACCINE COMPRISING INACTIVATED REOVIRUS-LIKE CALF-DIARRHEA VIRUS AND A CARRIER OR ADJUVANT THEREFOR.

United States Patent 3,839,556 CALF DIARRHEA VIRUS VACCINE AND RROCESSESCharles A. Mebus and Marvin J. Twiehaus, Lincoln, Nebr., assignors toBoard of Regents of the University of Nebraska, Lincoln, Nebr.

No Drawing. Continuation-impart of application Ser. No.

197,520, Nov. 10, 1971, which is a continuation-in-part ABSTRACT OF THEDISCLOSURE Calf diarrhea virus vaccines are prepared by passaging calfdiarrhea virus obtained from feces of infected calves in tissue culture,particularly bovine or swine tissue culture, from one to about twohundred and fifty times. Inactivated vaccines are also prepared.

This application is a continuation-in-part of copending application Ser.No. 197,520, filed Nov. 10, 1971, which application was acontinuation-in-part of application Ser. No. 90,481, filed Nov. 17,1970, now abandoned, which application was a continuation-in-part ofapplication Ser. No. 880,956, filed Nov. 28, 1969, now abandoned.

This invention relates to calf diarrhea virus vaccines, methods ofpreparing said vaccines, and methods of using them.

In particular, the invention comprises a process of pre paring calfdiarrhea virus vaccines by passaging calf diarrhea virus in tissueculture, particularly, but not limited to, bovine or swine tissueculture.

The invention also comprises a method of immunizing newborn calvesagainst calf diarrhea by administering to pregnant cows prior to thebirth of said calves a vaccine prepared by passaging calf diarrhea virusin tissue culture up to about 250 times.

The invention further comprises a method of immunizing newborn calvesagainst calf diarrhea by administering to said calves a vaccine preparedby passaging calf diarrhea virus in tissue culture up to about 250times.

The invention also comprises the vaccines, when produced by theinventive methods.

Neonatal calf diarrhea, also known as calf scours or calf enteritis, isa serious, contagious disease of newborn calves. There have beennumerous reports in the literature starting that E. coli is the cause ofneonatal calf diarrhea. However, this disease cannot consistently bereproduced with E. coli isolated from cases of neonatal diarrhea. Inaddition, Smith and Halls, J. Path. and Bact. 93, 499-529 1967),concluded that E. coli had not played an eitological role in 120 of 127epidemiologically unrelated cases of diarrhea in colostrum-fed calves invarious parts of the world.

Recently, Mebus et al. (University of Nebraska Research Bulletin 233,March 1969) have shown that nenonatal calf diarrhea is a clinical entitycaused by a specific Reo-like virus. Consistent experimentalreproduction of the disease verifying this finding was initiallyaccomplished by inoculating feces collected from neonatal calf diarrheafield cases via a duodenal cannula into a newborn caughtcolostrum-deprived calf. This calf developed diarrhea 15 hourspostinoculation. Its feces were collected and inoculated via a duodenalcannula into a hysterectomy-derived colostrum-deprived calf. Diarrheticfeces from this calf was passed in a similar manner in anotherhysterectomy-derived colostrum-deprived calf.

Patented Oct. 1 1974 Third, fourth and fifth virus passages in calveswere accomplished using bacteria-free fecal filtrates inoculated viaduodenal cannulas. Three calves in an isolation unit wrere inoculatedorally with bacteria-free filtrates of the 6th calf passage of virus,and all were free of E. coli before and throughout the diarrheticperiod. A seventh calf passage using feces from one of these E.coli-free calves caused diarrhea 16 hours postinoculation.

Immunifluorescent staining (as described by Mebus et al., supra) ofsections of small intestine from seventh passage calves revealed brightfluorescence in epithelial cells of the villi. Applying theimmunofiuorescent technique to dried fecal smears of field cases ofneonatal calf diarrhea, this virus has been found in herds in Nebraskaand in several other states.

Fluorescence was not observed when the sections were stained with hogcholera or bovine virus diarrhea conjugates. The viral etiology of calfdiarrhea virus therefore has now been clearly established.

The vaccines of the present invention accordingly are prepared fromvirus obtained by collecting feces of infected calves and filtering. Thefeces to be used are preferably collected from calves which have beenpreviously inoculated with a bacteria-free filtrate of feces containingfluorescing cells. Passing the virus through one or more calvesincreases the virus titer in the collected feces and thereby aidssubsequent growth of the virus in tissue culture for preparation ofvaccine. If the virus originally obtained from feces will grow on tissueculture without further passaging in the calf, this virus material maybe used. The feces so collected are diluted with phosphatebufferedsaline, and the mixture is centrifuged at 1000 g. for about 30 minutesand filtered through a suitable bacteriologic filter. The filtratecontaining virus then can be added to tissue culture for preparation ofvaccine.

The tissue culture in which the virus is passaged include a variety ofprimary cells and cell lines, although bovine and swine cells arepreferred. Among these materials are primary fetal bovine kidney cells,primary fetal bovine lung cells, primary fetal bovine thyroid cells,primary porcine kidney cells, primary fetal bovine choroid plexes cells,primary lamb kidney cells, bovine kidney cell line BK 1A, embryonicbovine trachea cell line EBTr (ATCC No. CCL 44), baby hamster kidneyclone 21 cell line BHK (ATCC No. CCL 10), Madin-Darby canine kidney cellline (ATCC No. CCL 22), green monkey kidney cell line VERO (ATCC No. CCL81), pig kidney cell line PK-15 (ATCC No. CCL 33), swine testicle cellline ST (available from the National Animal Disease Laboratory, Ames,Iowa), and Henles intestine (human embryo) cell lines HI, Hela, andLLC-MK (ATCC No. Nos. CCL, 6, 2, and 7, respectively). All the abovesubstrates support to some degree the growth of the virus, althoughother primary cells and cell lines may also be used. The primary cellsare obtained from healthy animals and grown according to standardmethods well known to the art. The cell lines are either readilyavailable from public depositories or may be grown and developed byknown techniques.

In order to prepare the vaccines of this invention, the virus is firstattenuated by passage in tissue culture. The cells are grown in asuitable growth medium. The cell sheets are then washed with aserum-free balanced salt solution. Virus is either added and allowed toadsorb onto the cells or is added directly to a suitable maintenancemedium, which is in turn added to the cells. The cells are thenincubated at a temperature of from 20-40 C., 28- 38 being preferred.Growth of virus is permitted to continue until a noticeablecytopathologic effect is observed. This occurs within 2 to 10 days, andthe medium containing the virus is then transferred to a new bottlecontaining tissue culture for additional passaging.

Choice of maintenance media is not critical and is within the normalskill of the art, except that it must not contain any material whichinhibits virus growth. A serumfree maintenance medium is preferred.Among those which are suitable are Earlcs or Hanks balanced saltsolutions containing 0.5% lactalbumin, 0.1% yeast extract, penicillin,and streptomycin, as well as Earles or Hanks base 199, 0.5 lactalbuminhydrolyzate, penicillin, and streptomycin.

Cytopathologic effect is apparent from the flagging produced, butinfection is also followed by immunofluorescence. The virus passages maybe conducted in stationary or roller bottle cultures. Use of high puritywater in the culture media may be required.

At the conclusion of each passage, the medium and cells are harvestedand then added to additional cells for further passaging. Subsequentpassages are conducted, if needed, to obtain a suitable vaccine. Thenumber of passages needed to prepare a suitable vaccine is determined byinoculating a vaccine of a particular passage level into susceptiblecalves. The desired passage level is one that itself causes no clinicalillness when inoculated orally into a newborn colostrum-deprived calfand prevents illness when the calf is subsequently inoculated with abacteriafree filtrate of feces containing virulent virus. The numberwill generally be from about 37 to about 250, 80-250 being preferred.

When the virus has been suitably attenuated, it may be propagated bygrowth in tissue culture. Virus is either added and allowed to adsorbonto the cells or is added directly to a suitable maintenance medium,preferably serum-free, which is then added to the cells. The cells areincubated and growth allowed to continue at 2040 until a suitable viraltiter, e.g. at least 10 and preferably up to about 10 or greater, isobtained.

The attenuated live vaccine may be used in the liquid state, the liquidmedium being the carrier, or it may be lyophilized and laterreconstituted for inoculation with a suitable vehicle or carrier, suchas sterile water. It may be administered to a cow or calf either orallyor parenterally.

When directly immunizing neonatal calves, an attenuated or modified livevaccine should be used, one suflicient to create immunity withoutcausing illness; such a vaccine will require up to about 250 passages.For optimum results, the calves should be immunized shortly after birth,i.e. before they have been exposed to virulent virus. This is preferablywithin the first day of life. A dose of 4 ml. of lyophilized andreconstituted virus containing approximately TCID/ml. is suitable. Thisdose containing 10 to 10 TCID/ml. is acceptable.

When immunizing calves by administering vaccine to the pregnant mother,the vaccine should be one with the same titer. The preferred dose shouldcontain the same amount of virus as is preferred for the calf, but ismade up to 2 ml. The vaccine should be administered to the cowparenterally, preferably subcutaneously or intramuscularly, to 90 daysprior to calving. Two doses are preferred, one 60 to 90 days beforecalving, the second 30 to 60 days before calving.

The invention also includes inactivated vaccines which are used toimmunize cows. This cow vaccine may be made by inactivating the livevirus by any known means. The titer of the virus should be as high aspossible within the range of 10 to 10 TCID/mL, or even higher; any cellpassage level of virus may be used, even unattenuated virus. Theinactivation may be carried out chemically, as with, for example,formalin or propiolactone, or with ultraviolet radiation or heat, forsuch a length of time and concentration as to effectively inactivate thevirus. These details are known to the art. Any known adjuvant may beadded for enhancement of antigenicity. Such adjuvants may includealuminum hydroxide gel, potassium alum, alginate, or an oil base orother adjuvant such as mineral or other organic oil. The inactivatedvaccine EXAMPLE 1 Primary fetal bovine lung cells, obtained from 89month bovine fetuses by well-known techniques, were grown in Roux flaskscontaining Earles lactalbumin hydrolyzate and 10% calf serum. Prior toinoculation, the cell monolayers were washed 2 or 3 times with Hanksbalanced salt solution. Following inoculation with calf passage fecalfiltrate, the virus was allowed to adsorb at 37 C. for two hours, withrocking of the bottle every half hour to redistribute the inoculum.Maintenance medium, consisting of Earles balanced salt solutioncontaining 0.5% lactalbumin and 0.1% yeast extract, plus penicillin andstreptomycin, was added. The cells were then incubated at 37 C. untilthe sheet showed cytopathogenic effect or fluorescence with specificantibodies.

When to of cells in the monolayer fluoresced, the cells and fluid wereharvested by freezing and thawing. The volume was reduced by 50% in anultracentrifuge. The cells and fluid were stored at 60 C.

For evaluation of the vaccine, this cell-fluid mixture was thawed andtested both as such and mixed with an equal quantity of a safflower oilbase adjuvant. The vaccine was injected intramuscularly into pregnantcows 60 days prior to calving. Eight cows were injected with theabove-described tissue culture fluid, 7 cows were injected with the sameamount of tissue culture fluid mixed with an equal quantity of adjuvant,and 6 cows were injected with 5 m1. of adjuvant mixed with 5 ml. of aviral suspension prepared from feces of experimental calves withneonatal diarrhea, as described by Mebus et al., as noted above for thepreparation of antigen. Thirty-nine cows were left as nonvaccinatedcontrols. The cows were sent to calve on a ranch where neonatal calfdiarrhea had been a problem for several years and was then occurring.The eflicacy of the vaccine was determined by observing the calves fordiarrhea and testing the feces of the calves for presence of virus ifdiarrhea occurred. The virus was identified by immunofluorescence. Theresults are set forth in Table 1.

TABLE l.-Efiicacy of Various Vaccines Against Calf Diarrhea All controlcalves developed diarrhea; 7 of 8 calves from tissue culture-vaccinatedcows developed diarrhea; 4 of 6 calves from fecal virus plusadjuvant-vaccinated cows developed diarrhea and 3 of 7 calves fromtissue culture plus adjuvant-vaccinated cows developed diarrhea. It isapparent that all the vaccines had some eflicacy, even in the face of asevere, possibly overwhelming, challenge of infection. The best resultswere obtained with the use of adjuvanted tissue culture fluid and cells.

On the basis of these results, the tissue culture plus adjuvant vaccinewas evaluated in a dairy herd that had had a severe neonatal calfdiarrhea problem for two years and in which the virus had also beendetected by immunofluorescence. Cows were selected for vaccination sothere would be 5 vaccinated cows per week calving. They were vaccinatedat 30 day intervals 3090 days prior to parturition. Colostrum fromvaccinated cows collected 0-5 days postcalving was then fed to calves0-13 days old. Milk from vaccinated cows collected 6-10 days postcalving was fed to calves 14 to 28 days old. The procedure permitted theevaluation of the interval required between vaccination and calving aswell as effect of 2 doses of vaccine.

The data are summarized in Tables 2 and 3. In Table 2, the incidence ofdiarrhea in calves which received colostrum from non-vaccinated cows andfrom cows which received one dose of vaccine is tabulated. In Table 3,the incidence of diarrhea in calves which received colostrum from cowsthat had received two doses of vaccine is tabulated.

TABLE 2.Incidence of Calf Diarrhea in Animals Born in Cows Unvaccinatedor Vaccinated Once No. calves 180 No. of diarrhetic calves 87 (48.3%)

TABLE 3.Incidence of Calf Diarrheain Animals Born of Cows VaccinatedTwice No. calves 253 Dates of birth 8/30-10/24 No. of diarrhetic calves69 (27.3%)

It can be seen from these results that the incidence of neonatal calfdiarrhea was reduced from 48.3% in 180 calves to 27.3% in 253 calves byvaccination more than once.

Further improvement should be obtainable by ensuring that all new-borncalves are fed colostrum from vaccinated cows as soon as possible afterbirth. In the above experiment, for unavoidable reasons, up to 12 hourselapsed before some calves were fed. The disease thus might have beenestablished already in some animals before receiving the colostrum.

In the above experiments, no adverse effect was observed in any of thevaccinated animals.

EXAMPLE 2 Other vaccines for administration to pregnant cows areprepared by passaging virus which has been adapted to grow in bovine,swine, or other primary cell or cell line tissue cultures by priorpassaging, if necessary, in calves. The number of passages for asuitable vaccine is that necessary to obtain a virus which does notcause disease in calves, but induces immunity.

EXAMPLE 3 A vaccine for direct immunization of calves was prepared bypassaging the virus in cells of the pig kidney cell line PK-IS (ATCC No.CCL 33) 37 times according to the procedure of Example 1. The passageswere conducted in stationary tubes.

EXAMPLE 4 Other calf vaccines can be prepared by passaging virus whichhas been adapted to grow in bovine, swine, or other primary cell or cellline tissue cultures by prior passaging, if necessary, in calves. Thenumber of passages for a suitable vaccine is determined by testingmaterial for its infectious and antigenic properties.

EXAMPLE 5 Fecal material ml.) is collected from field cases of neonatalcalf diarrhea and then refrigerated or quickfrozen. The feces arescreened by the fluorescent antibody method or electron microscopy forthe presence of virus. The material containing virus is diluted with athreefold volume of phosphate buffered saline, the mixture iscentrifuged and put through a bacteriological filter. A 5 Seitz filter,then a La, and then a 0.5 filter are used. Then ml. is inoculated intothe duodenum of a calf. Instead of filtering the material, the wholefeces may be directly inoculated.

Feces are collected from the animal as soon as diarrhea has begun. Thefeces are treated in the same manner as above and passed via duodenalinjection two or more times. After each calf passage, a filtrate isprepared and inoculated onto primary bovine kidney cells or primarybovine lung cells in stationary or rolling culture tubes or bottles. Upto 4 passages are made in these cells, the cells being examined for aflagging-type cytopathologic effect and monitored by the fluorescentantibody method. If neither is observed, the cells are discarded. Ifvirus is detected, further passages are conducted.

EXAMPLE 6 The virus was passaged 40 times at 37 in PK15 pig kidneycells. It was then passaged 135 more times at 37 in primary bovinekidney cells. For the bovine kidney passages, the virus at first waspassed every 3 or 4 days by inoculating an 8 oz. bottle with 1 ml. ofinfected tissue culture fluid. Prior to inoculation, the cells werewashed 3 times with Hanks balanced salt solution. At first virus wasadsorbed for 2 hours before maintenance medium was put on, and later foronly 1 hour. After about the 70th bovine kidney passage, the virus waspassed every day. Beginning with the 120th passage, the virus was notabsorbed. The bottles were washed, maintenance medium was then added,and the cells inoculated with the virus fluid.

Tissue culture fluid from the th to the 175th total passage level,harvested 5 days after inoculation, has been mixed with an equalquantity of safllower oil base adjuvant and inoculated into pregnantcows. The incidence of diarrhea in calves from these cows was reducedabout 50% and mortality almost eliminated.

EXAMPLE 7 The virus was passaged 40 times at 37 in PK-l5 pig kidneycells, times at 37 in primary bovine kidney cells, and 17 times at 31 inprimary bovine 'kidney cells. The resulting tissue culture fluid, whenharvested and inoculated (12 ml.) into the duodenum of aCaesariansection colostrum-deprived calf, caused no illness.

EXAMPLE 8 Fecal material from field cases of neonatal calf diarrhea wasprocessed as in Example 5 and passed through 2 duodenal passages. Theresulting fecal filtrate was then passaged in roller tubes containingprimary bovine kidney cells. Stationary bottles were used starting withthe 3rd passage. 163 passages were made at 37 and the resulting tissueculture fluid containing virus, when combined with adjuvant and injectedinto pregnant cows, effectively protected the calves from disease-causedmortality and greatly lowered the incidence of diarrhea.

EXAMPLE 9 Tissue culture fluid containing virus from 114 passages at 37was then passed 16 more times at 31. The fluid was injected into calves4 hours old. When the calves were challenged with virulent virus when 28hours old, they did not develop illness.

EXAMPLE 10 A vaccine may be prepared by duodenally passaging fecalmaterial from diarrhetic calves from 1 to 4 times in calves and thenpassaging the resulting fecal 'material in a swine testicle cell lineuntil the tissue culture fluid, when injected duodenally into calves, nolonger produces illness.

EXAMPLE 11 Fecal material from field cases of neonatal calf diarrhea wasprocessed as in Example 5 and passed through 2 duodenal bacteria-freecalf passages. The resulting fecal filtrate was then passaged in primarybovine kidney tissue culture 114 times at 37 and 60 times at 30. It wasfurther passaged 20 times at 37 in a bovine kidney cell line to achievesuitable attenuation.

The virus in the resulting tissue culture fluid is propagated in thebovine kidney cell line at about 36 to obtain a suitable virus titer andthen harvested.

The virus from the 174th primary bovine kidney passage, afterpropagation in the cell line, was lyophilized and reconstituted and usedin 4 ml. doses containing TCID/ml. on calves 6 to 7 hours old.

The above-obtained vaccine has been used successfully in experimentalhysterectomy-derived, colostrum-deprived calves and in ranch herds. Thegreat majority of the experimental calves remained clinically normalafter vaccination and following challenge with virulent virus. In theherds, vaccination significantly reduced the incidence of calf diarrheaand death in the herds infested with reovirus.

EXAMPLE l2 Inactivated vaccines were prepared both from virulent viruspropagated on primary fetal bovine kidney cells and from the attenuatedvirus as obtained in Example 11 by adding sufficient formalin to make a0.2% final concentration and incubating the mixture at 37 for 48-72hours. Equal volumes of formalinized cell culture fluid and an oil baseadjuvant (safflower oil emulsion) were emulsified in a blender.

Doses of 3 to 10 ml. of these vaccines were administered to pregnantranch cows 30 to 90 days prior to calving. The incidence of calfdiarrhea and death from the reoviruslike agent were significantlyreduced.

We claim:

1. An inactivated reovirus-like calf-diarrhea virus vaccine comprisinginactivated reovirus-like calf-diarrhea virus and a carrier or adjuvanttherefor.

2. An inactivated reovirus-like calf-diarrhea virus vaccine according toclaim 1, in which the virus has been inactivated with formalin.

3. An inactivated reovirus-like calf-diarrhea virus vaccine according toclaim 1, in which the adjuvant is safflower oil.

4. A method of immunizing calves against reovirus-like calf diarrheacomprising parenterally administering to a pregnant cow carrying saidcalf from to days prior to calving an inactivated reovirus-like calfdiarrhea vaccine as claimed in claim 1.

5. A method of immunizing calves against reovirus-like calf diarrheacomprising parenterally administering to a pregnant cow carrying saidcalf from 30 to 90 days prior to calving an inactivated reovirus-likecalf diarrhea vaccine as claimed in claim 2.

6. A method of immunizing calves against reovirus-like calf diarrheacomprising parenterally administering to a pregnant cow carrying saidcalf from 30 to 90 days prior to calving an inactivated reovirus-likecalf diarrhea vaccine as claimed in claim 3.

References Cited UNITED STATES PATENTS 3,293,129 12/1966 Baker 42489OTHER REFERENCES Mebus et al., University of Nebraska College of Agric.& Home Econ, Res. Ball. 233, March 1969.

Mebus et al., Proc. 73rd Ann. Mtg. U .S. Amimal Health Assoc., Oct.12-17, 1969, pp. 97-99.

Welch, Canad. J. Comp. Med. 35; 195-202 (1971).

Mebus et al., Canad. Vet. Journal 12; 69-72 (1971).

Mebus et al., Vet. Med/Small Anim. C1in., 67; 173-178 (1972).

Stair et al., Amer. J. Vet. Res. 33,- 1147-1155 (1972).

Mebus et al., Amer. J. Vet. Res. 34; -150 (1973).

Mebus et al., Vet Path. II; 375 (1973).

Mebus et al., I. Am. Vet. Med. Assoc. 163 (7); 880-883 (1973).

'SHEP K. ROSE, Primary Examiner

1. AN INACTIVATED REOVIRUS-LIKE CALF-DIARRHEA VIRUS VACCINE COMPRISINGINACTIVATED REOVIRUS-LIKE CALF-DIARRHEA VIRUS AND A CARRIER OR ADJUVANTTHEREFOR.